Cynthia Czajkowski

Structural requirements for eszopiclone and zolpidem binding to the gamma-aminobutyric acid type-A (GABAA) receptor are different.

The sleep-aids zolpidem and eszopiclone exert their effects by binding to and modulating gamma-aminobutyric acid type-A receptors (GABA(A)Rs), but little is known about the structural requirements for their actions. We made 24 cysteine mutations in the benzodiazepine (BZD) binding site of alpha(1)beta(2)gamma(2) GABA(A)Rs and measured zolpidem, eszopiclone, and BZD-site antagonist binding. Mutations in gamma(2)loop D and alpha(1)loops A and B altered the affinity of all ligands tested, indicating that these loops are important for BZD pocket structural integrity. In contrast, gamma(2)loop E and alpha(1)loop C mutations differentially affected ligand affinity, suggesting that these loops are important for ligand selectivity. In agreement with our mutagenesis data, eszopiclone docking yielded a single model stabilized by several hydrogen bonds. Zolpidem docking yielded three equally populated orientations with few polar interactions, suggesting that unlike eszopiclone, zolpidem relies more on shape recognition of the binding pocket than on specific residue interactions and may explain why zolpidem is highly alpha(1)- and gamma(2)-subunit selective.

The relative amount of cRNA coding for γ2 subunits affects stimulation by benzodiazepines in GABAA receptors expressed in Xenopus oocytes

Benzodiazepine (BZD) potentiation of GABA-activated Cl(-)-current (I(GABA)) in recombinant GABA(A) receptors requires the presence of the gamma subunit. When alpha1, beta2 and gamma2S cRNA are expressed in a 1:1:1 ratio in Xenopus oocytes, BZD potentiation of I(GABA) is submaximal, variable and diminishes over time. Potentiation by BZDs is increased, more reproducible and is stabilized over time by increasing the relative amount of cRNA coding for the gamma2S subunit. In addition, GABA EC(50) values for alpha1beta2gamma2 (1:1:1) receptors are intermediate to values measured for alpha1beta2 (1:1) and alpha1beta2gamma2 (1:1:10) receptors. We conclude that co-expression of equal ratios of alpha1, beta2 and gamma2 subunits in Xenopus oocytes produces a mixed population of alpha1beta2 and alpha1beta2gamma2 receptors. Therefore, for accurate measurements of BZD potentiation it is necessary to inject a higher ratio of gamma2 subunit cRNA relative to alpha1 and beta2 cRNA. This results in a purer population of alpha1beta2gamma2 receptors.

Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit mutation [γ2(R43Q)] found in human epilepsy

The γ-aminobutyric acid type A (GABAA) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABAA receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABAA γ2-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABAA receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from α1β2γ2 or α1β2γ2(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imidazopyridine zolpidem. These results provide evidence of impaired GABAA receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Structural mechanisms underlying benzodiazepine modulation of the GABAA receptor

Many clinically important drugs target ligand-gated ion channels; however, the mechanisms by which these drugs modulate channel function remain elusive. Benzodiazepines (BZDs), anesthetics, and barbiturates exert their CNS actions by binding to GABAA receptors and modulating their function. The structural mechanisms by which BZD binding is transduced to potentiation or inhibition of GABA-induced current (IGABA) are essentially unknown. Here, we explored the role of the γ2Q182-R197 region (Loop F/9) in the modulation of IGABA by positive (flurazepam, zolpidem) and negative [3-carbomethoxy-4-ethyl-6,7-dimethoxy-β-carboline (DMCM)] BZD ligands. Each residue was individually mutated to cysteine, coexpressed with wild-type α1 and β2 subunits in Xenopus oocytes, and analyzed using two-electrode voltage clamp. Individual mutations differentially affected BZD modulation of IGABA. Mutations affecting positive modulation span the length of this region, whereas γ2W183C at the beginning of Loop F was the only mutation that adversely affected DMCM inhibition. Radioligand binding experiments demonstrate that mutations in this region do not alter BZD binding, indicating that the observed changes in modulation result from changes in BZD efficacy. Flurazepam and zolpidem significantly slowed covalent modification of γ2R197C, whereas DMCM, GABA, and the allosteric modulator pentobarbital had no effects, demonstrating that γ2Loop F is a specific transducer of positive BZD modulator binding. Therefore, γ2Loop F plays a key role in defining BZD efficacy and is part of the allosteric pathway allowing positive BZD modulator-induced structural changes at the BZD binding site to propagate through the protein to the channel domain.

An Arginine Involved in GABA Binding and Unbinding But Not Gating of the GABAA Receptor

GABAA receptor function can be conceptually divided into interactions between ligand and receptor (binding) and the opening and closing of the ligand-bound channel (gating). The relationship between binding, gating, and receptor structure remains unclear. Studies of mutations have identified many amino acid residues that contribute to the GABAbinding site. Most of these studies assayed changes in GABA dose–response curves, which are macroscopic measures that depend on the interplay of many processes and cannot resolve individual microscopic transitions. Understanding the microscopic basis of binding and gating is critical, because kinetic transition rates predict how receptors will behave at synapses. Furthermore, microscopic rates are directly related to the molecular interactions underlying receptor function. Here, we focused on a residue (β2-R207) previously identified as lining the GABA-binding site that, when mutated to cysteine, greatly reduces apparent GABA affinity and was predicted to affect both binding and gating. To better understand the role of β2-R207, we expressed α1β2 and α1β2-R207C receptors in human embryonic kidney 293 cells and studied receptor kinetics using fast solution applications. The mutation accelerated deactivation by 10-fold, without altering desensitization in the presence of saturating GABA. Maximum open probability and single-channel open times were also unaltered by the mutation, but the GABA-binding rate was reduced eightfold. Therefore, the effects of this mutation in a predicted binding site residue are solely attributable to changes in GABA-binding and unbinding kinetics, with no changes in channel gating. Because β2-R207 stabilizes GABA in the binding pocket, it may directly contact the GABA molecule.

Mutation of Glutamate 155 of the GABAA Receptor β2 Subunit Produces a Spontaneously Open Channel: A Trigger for Channel Activation

Protein movements underlying ligand-gated ion channel activation are poorly understood. The binding of agonist initiates a series of conformational movements that ultimately lead to the opening of the ion channel pore. Although little is known about local movements within the GABA-binding site, a recent structural model of the GABAA receptor (GABAAR) ligand-binding domain predicts that β2Glu155 is a key residue for direct interactions with the neurotransmitter (Cromer et al., 2002). To elucidate the role of the β2Ile154-Asp163 region in GABAAR activation, each residue was individually mutated to cysteine and coexpressed with wild-type α1 subunits in Xenopus laevis oocytes. Seven mutations increased the GABA EC50 value (8- to 3400-fold), whereas three mutations (E155C, S156C, and G158C) also significantly increased the 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium (SR-95531) KI value. GABA, SR-95531, and pentobarbital slowed N-biotinylaminoethyl methanethiosulfonate modification of T160C and D163C, indicating that β2Thr160 and β2Asp163 are located in or near the GABA-binding site and that this region undergoes structural rearrangements during channel gating. Cysteine substitution of β2Glu155 resulted in spontaneously open GABAARs and differentially decreased the GABA, piperidine-4-sulfonic acid (partial agonist), and SR-95531 sensitivities, indicating that the mutation perturbs ligand binding as well as channel gating. Tethering thiol-reactive groups onto β2E155C closed the spontaneously open channels, suggesting that β2Glu155 is a control element involved in coupling ligand binding to channel gating. Structural modeling suggests that the β2 Ile154-Asp163 region is a protein hinge that forms a network of interconnections that couples binding site movements to the cascade of events leading to channel opening.

A conserved salt bridge critical for GABAA receptor function and loop C dynamics

Chemical signaling in the brain involves rapid opening and closing of ligand gated ion channels (LGICs). LGICs are allosteric membrane proteins that transition between multiple conformational states (closed, open, and desensitized) in response to ligand binding. While structural models of cys-loop LGICs have been recently developed, our understanding of the protein movements underlying these conformational transitions is limited. Neurotransmitter binding is believed to initiate an inward capping movement of the loop C region of the ligand-binding site, which ultimately triggers channel gating. Here, we identify a critical intrasubunit salt bridge between conserved charged residues (βE153, βK196) in the GABAA receptor (GABAAR) that is involved in regulating loop C position. Charge reversals (E153K, K196E) increased the EC50 for GABA and for the allosteric activators pentobarbital (PB) and propofol indicating that these residues are critical for channel activation, and charge swap (E153K-K196E) significantly rescued receptor function suggesting a functional electrostatic interaction. Mutant cycle analysis of alanine substitutions indicated that E153 and K196 are energetically coupled. By monitoring disulfide bond formation between cysteines substituted at these positions (E153C-K196C), we probed the mobility of loop C in resting and ligand-bound states. Disulfide bond formation was significantly reduced in the presence of GABA or PB suggesting that agonist activation of the GABAAR proceeds via restricting loop C mobility.

Charged Residues in the α1 and β2 Pre-M1 Regions Involved in GABAA Receptor Activation

For Cys-loop ligand-gated ion channels (LGIC), the protein movements that couple neurotransmitter binding to channel gating are not well known. The pre-M1 region, which links the extracellular agonist-binding domain to the channel-containing transmembrane domain, is in an ideal position to transduce binding site movements to gating movements. A cluster of cationic residues in this region is observed in all LGIC subunits, and in particular, an arginine residue is absolutely conserved. We mutated charged pre-M1 residues in the GABAA receptor α1 (K219, R220, K221) and β2 (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type β2 or α1 in Xenopus oocytes. Cysteine substitution of β2R216 abolished channel gating by GABA without altering the binding of the GABA agonist [3H]muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto α1K219C, β2K213C, and β2K215C increased maximal GABA-activated currents, suggesting that structural perturbations of the pre-M1 regions affect channel gating. GABA altered the rates of sulfhydryl modification of α1K219C, β2K213C, and β2K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified β2K213C and β2K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of β2K215C by the negatively charged reagent. Overall, the data indicate that the pre-M1 region is part of the structural machinery coupling GABA binding to gating and that the transduction of binding site movements to channel movements is mediated, in part, by electrostatic interactions.

Effects of γ2S subunit incorporation on GABAA receptor macroscopic kinetics

Abstract GABAA receptors, the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are heteropentameric proteins. We are interested in understanding the contribution of the γ subunit to the kinetic properties of GABAA receptors. Studies in Xenopus oocytes have suggested that co-expression of α1, β2, and γ2S subunits results in the formation of both αβ and αβγ receptors ( Boileau et al., 2002a , Boileau et al., 1998 ). Here, we have used an excess of the γ2S subunit in transfections of HEK293 cells to bias expression toward αβγ-containing receptors. Using rapid application and whole cell patch clamp techniques, we found that incorporation of the γ subunit eliminated the rapid phases of desensitization and accelerated deactivation, consistent with a proposed role of desensitization in slowing deactivation. In addition, αβγ receptors had an increased GABA EC50, reduced sensitivity to block by Zn2+, and did not display outward rectification as compared to αβ receptors.

Tandem subunits effectively constrain GABAA receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAA receptor-associated protein.

GABAergic synapses likely contain multiple GABAA receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of α, β, and γ subunits can be confounded by receptors expressed from α and β subunits alone. To produce recombinant GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a “tandem” α1 subunit linked to a β2 subunit. Cotransfection of the γ2 subunit with αβ-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the γ subunit with nonlinked α1 and β2 subunits. Similarly, expression of α subunits together with αβ-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of α with β subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of αβ-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of α1β2 and α1β2γ2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in α1β2γ2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of αβγ receptors.